PROJECT SUMMARY The treatment of neuropathic pain (NP) remains unsatisfactory and presents a major global health and economic burden to individuals and society. Unfortunately, currently available medications are not uniformly effective and are associated with significant central nervous system (CNS) side effects that limit their clinical utility. Translation from preclinical findings to a viable clinical therapy for NP has been fraught with disappointments, possibly because animal studies have primarily relied on outcome measures such as reflex responses that fail to mimic the chief symptom of spontaneous and ongoing pain in patients. Our recent studies suggested that peripherally acting -opioid receptor (MO-R) agonists alleviate mechanical and heat hypersensitivities in rodent models of NP. Here, our preliminary studies also indicate a role for peripheral cannabinoid receptors (CB-R) in NP, and further suggest that both peripheral MO-R and CB-R are valuable targets for alleviating ongoing pain after nerve injury. Based on these novel findings, we hypothesize that: 1) Activation of CB1-R and MO-R in the peripheral nervous system (PNS) attenuates ongoing NP, and has no significant adverse effects that would suggest CNS penetration, impaired G.I. motility, or undesirable immune effects; 2) Subpopulations of DRG neurons, especially those that express CB1-R, MO-R, and TRPV1, develop spontaneous activity after injury that can be inhibited by peripherally acting CB1-R and MO-R agonists; 3) Activation of peripheral neuronal CB1-R and MO-R decreases nociceptive transmission in dorsal horn neurons; 4) CB1-R agonists enhance peripherally acting MO-R agonist-induced inhibition of NP-related behavior by potentiating MO-R-mediated inhibition of DRG neurons. A broad range of strategies will be used to rigorously test our hypotheses in three inter-related Aims. In Aim 1, the efficacy, receptor subtypes involved, and potential adverse effects of systemic administration of the drugs will be studied. In Aim 2, we will use calcium imaging, patch clamp recording in spinal cord slices, and in vivo dorsal horn recordings to determine the mechanisms by which CB13 and DALDA inhibit ongoing NP in the PNS. In Aim 3, we will examine the functional interactions between CB1-R and MO-R in PNS for NP inhibition. Our study involves comprehensive and innovative approaches including: Operant behavioral tests to reveal the relief of ongoing and affective components of NP, high-throughput Pirt-GCaMP6 calcium imaging of DRG neurons in vivo, peripherally restricted CB1-R and MO- R conditional knockout (cKO) mice using a Cre/loxP system, neurophysiologic recordings in functionally distinct subgroups of dorsal horn neurons (e.g., excitatory, GABAergic inhibitory) identified by fluorescence, and multiplex cytokine ELISA. The results of these studies will provide new insights into the mechanisms that underlie the peripheral pain-inhibitory effects of opioids and cannabinoids, and will contribute to the development of novel, translatable therapeutic strategies for the treatment of NP that may be associated with minimal adverse effects. Accordingly, the scientific premise, innovation, and rigor of our proposal are high.